The biochemistry of apoptosis

JC-1 ANALYSIS OF OXIDATIVE STRESS

INTRODUCTION

The role of the mitochondria has been under investigation for the past 50 years especially its role in tumour cells. This is because the researchers lacked the appropriate technique to investigate these organelles that are present in living cells. About three decades ago, fluorescent cationic dyes and also flow cytometry were used to investigate the functions and structure of the mitochondria (Ratinaud et al, 1988; Petit, 1992). In the course of a lot of pathological and physiological processes, mitochondrial transmembrane potential mt alterations may talk place (Sobreira et al, 1996; Skowronek et al, 1992). This is of utmost importance because these organelles have been implicated in apoptosis (Henkart and Grinstein, 1996; Green and Reed, 1998). The mitochondrion plays a significant role in cell death and changes signify derogatory marker in the process which could be at the extremes of apoptosis (Hirsch et al, 1998). The disruption of the mitochondrial integrity and the loss of mitochondrial transmembrane potential is as a consequence of the mitochondrial permeability transition. (Castedo et al, 2002)These changes cause the mitochondria to eject organelles protein such as cytochrome c and others which eventually lead to the activation of caspase (Leist, 2001)

Flow cytometry with lipophilic dyes are methods used to quantify mitochondrial transmembrane potential ( Darzynkiewicz et al, 1982). Such dyes include DiOC6 ,Rh 123 or rhodamine 123 ( Ratinaud et al,1988) and JC-1( Cossarizza et al, 1993 ). These dyes are most popular in detecting small changes related to apoptosis in the functions mitochondria (Vermes et al, 2000). The Rh123 was later discovered to exhibit an increase in fluorescence after apoptosis. This was thought to be due to the mitochondrial swelling. (Metivier et al, 1998)

A more sensitive dye which was thought to accurately ascertain alterations in the activities of the mitochondria ( Cossarizza et al, 1993)The JC-1 which is lipophilic, cationic dye can selectively enter mitochondria in its monometric state and emits at 527nm after being excited at 488nm. As the membrane potential increases, the colour is reversed from green to red. It does this in healthy cells by that have a high mitochondrial membrane potential forming a complex referred to as the J-aggregates with great red fluorescence, emitting at 590nm.JC-1 stays in the monometric form when the ??m is low which is evident in unhealthy or apoptotic cells, hence a shift in fluorescence from red to green would be noticed.

The aim of this study is to generate and analyse data using fluorescence microscopy. Apoptosis was induced with varying concentrations of hydrogen peroxide or H202 and determination of the mitochondrial transmembrane potential was done with the JC-1 staining. Results were viewed under the confocal microscope.

MATERIALS AND METHODS

Chemicals

The chemical required for the experiment is the 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide or JC-1. To induce cell death, cells were placed in H2O2, tricaine, collagenase

Experimental procedures

(0.6 mg/ml) of tricaine was used to anaesthetise fish and then the fish was decapitated. L15 medium that contained 0.2g/L of collagenase was put in an Eppendorf tube and the dissected liver placed in the solution. The cells were then separated by the action of a pipette and then they were centrifuged at 12000 rpm for five seconds . The supernatant was then isolated and the pellet was extracted and employed in the experiment.

The liver cells were then put in three Eppendorf tubes and they were labelled as L, H and C ; (L- Low concentration of H2O2 ; H- High concentration of H2O2 and C- Control )

To the tube labelled L, 500 µl of 100 mM H2O2 in Hank's solution was added. To the tube labelled H, 500 µl of 1 M H2O2 in Hank's solution was put in and for control (C), 500 µl of Hank's solution was added.

Flow Cytometry

The potential of the mitochondrial membrane was then determined with the use of a fluorescence probe, the JC-1 (Ratinaud et al, 1988). The cells were then stained with 2 µl JC-1 solution. The tubes were enclosed with foils and left to stand at room temperature for thirty minutes.

For five seconds, the tubes were centrifuged at 12000 rpm using the Eppendorf centrifuge 5415C and the supernatants carefully removed thereafter from each tube to avoid mixing with the cells. The cells were resuspended in 100 µl Hanks solution.

Confocal analysis

50ul of the solution was then transferred to fresh glass slides labelled L, D and C and coverslips were placed on each one. The samples were then subjected to analysis with the epifluorescence microscope that has a confocal imaging system. Photographs were taken with the equipment and the images were produced. The effects of the various concentrations of H2O2were then studied carefully and notes taken

Results

The control cells (Fig A) treated with 500 µl of Hank's solution showed red fluorescence, an indication of viable cells present. Fig B treated with a concentration of 500 µl of 100 mM H2O2 in Hank's solution showed a larger population of orange cells, with some green stained cells. Fig C treated with a higher concentration of hydrogen peroxide (500 µl of 1 M H2O2 in Hank's solution) showed cells that display mainly green fluorescence possibly due to the formation of J- aggregates. Fig D showed another's group control of 500 µl of Hank's solution. It gave a better picture of cells to view under the confocal microscope. Fig A possibly had very few numbers of cells making it difficult to reveal a conclusive picture of its staining.

DISCUSSION

The major pathogenic agent of many diseases has been thought to be the mitochondria which are capable of producing reactive oxygen species. Trauma to cellular organelles and even oxidative stress can lead to ageing, cellular reduction in activities and apoptosis (Lenaz et al, 1999). Viral infections, reactive oxygen species, ultraviolet radiations (Henseleit et al, 1996) and a whole lot of extracellular stresses are known to induce apoptosis.(Mathiasen and Jaattela, 2002). Many individual pathways can bring about apoptosis of which the mitochondria is centrally involved and which is a signal for cellular damage haven occurred (Green and Reed, 1998; Henkart and Grienstein, 1996). When this happens, damage to the mitochondria brings about a fall in the mitochondria transmembrane potential which in turn leads to a release of organelle proteins (Castedo et al, 2002; Hirsch et al, 1998) and eventually the activation of caspase (Hengartner , 2000)

In order to differentiate between toxicity and effectiveness of a therapy, the study of necrosis against apoptosis is crucial (Zamai et al, 2001) Cell death can be studied by assessing the mt (Shapiro, 1994). The JC-1 has potentials in determining alterations in the mt even best, after oxidative injury, a common marker for the investigation of cell death. (Polla et al, 1996)

It was shown, according to the results that the levels of concentration of hydrogen peroxide, the cells can either die (Lelli et al, 1998) or remain viable. Concentration of H2O2 at 1 mM resulted in cell death hence the green fluorescence whereas with a concentration of 100 mM some cells were seen alive (red and green flouresence). Out of seven cells viewed under the confocal microscope, two were seen to fluorescence red, three orange and two appeared fluorescence green indicating that 100 mM concentration of H2O2 had little effect on the cells compared with the control. The number of cell death by apoptosis increased on exposure to 1 mM of H2O2

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